Purine Nucleoside Analog - Sulfinosine Modulates Diverse Mechanisms of Cancer Progression in...

Purine Nucleoside Analog - Sulfinosine ModulatesDiverse Mechanisms of Cancer Progression in Multi-DrugResistant Cancer Cell LinesMirjana Dacevic1, Aleksandra Isakovic1, Ana Podolski-Renic2, Andelka M. Isakovic1, Tijana Stankovic2,

Zorica Milosevic2, Ljubisav Rakic3, Sabera Ruzdijic2, Milica Pesic2*

1 Faculty of Medicine, University of Belgrade, Doktora Subotica 8, Belgrade, Serbia, 2 Institute for Biological Research, Department of Neurobiology, University of Belgrade,

Bulevar Despota Stefana 142, Belgrade, Serbia, 3 Serbian Academy of Sciences and Arts, Knez Mihailova 35, Belgrade, Serbia

Abstract

Achieving an effective treatment of cancer is difficult, particularly when resistance to conventional chemotherapy isdeveloped. P-glycoprotein (P-gp) activity governs multi-drug resistance (MDR) development in different cancer cell types.Identification of anti-cancer agents with the potential to kill cancer cells and at the same time inhibit MDR is important tointensify the search for novel therapeutic approaches. We examined the effects of sulfinosine (SF), a quite unexploredpurine nucleoside analog, in MDR (P-gp over-expressing) non-small cell lung carcinoma (NSCLC) and glioblastoma cell lines(NCI-H460/R and U87-TxR, respectively). SF showed the same efficacy against MDR cancer cell lines and their sensitivecounterparts. However, it was non-toxic for normal human keratinocytes (HaCaT). SF induced caspase-dependent apoptoticcell death and autophagy in MDR cancer cells. After SF application, reactive oxygen species (ROS) were generated andglutathione (GSH) concentration was decreased. The expression of key enzyme for GSH synthesis, gamma Glutamyl-cysteine-synthetase (cGCS) was decreased as well as the expression of gst-p mRNA. Consequently, SF significantly decreasedthe expression of hif-1a, mdr1 and vegf mRNAs even in hypoxic conditions. SF caused the inhibition of P-gp (coded by mdr1)expression and activity. The accumulation of standard chemotherapeutic agent – doxorubicin (DOX) was induced by SF inconcentration- and time-dependent manner. The best effect of SF was obtained after 72 h when it attained the effect ofknown P-gp inhibitors (Dex-verapamil and tariquidar). Accordingly, SF sensitized the resistant cancer cells to DOX insubsequent treatment. Furthermore, SF decreased the experssion of vascular endothelial growth factor (VEGF) on mRNAand protein level and modulated its secretion. In conclusion, the effects on P-gp (implicated in pharmacokinetics and MDR),GSH (implicated in detoxification) and VEGF (implicated in tumor-angiogenesis and progression) qualify SF as multi-potentanti-cancer agent, which use must be considered, in particular for resistant malignancies.

Citation: Dacevic M, Isakovic A, Podolski-Renic A, Isakovic AM, Stankovic T, et al. (2013) Purine Nucleoside Analog - Sulfinosine Modulates Diverse Mechanisms ofCancer Progression in Multi-Drug Resistant Cancer Cell Lines. PLoS ONE 8(1): e54044. doi:10.1371/journal.pone.0054044

Editor: Michihiko Kuwano, Kyushu University, Japan

Received July 26, 2012; Accepted December 5, 2012; Published January 11, 2013

Copyright: � 2013 Dacevic et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Ministry of Education, Science and Technological Development of Serbia (grant numbers III 41031 and III 41025) supported this research. The fundershad no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

Sulfinosine or SF (Figure 1, [R,S]-2-amino-9-b-D-ribofurano-

sylpurine-6-sulfinamide) is the oxidized form of 6-thioguanosine

[1]. It is a quite unexplored anti-cancer agent in comparison to

other thiopurines (6-thioguanine and 6-mercaptopurine). SF

inhibits cancer cell growth, at least partially, by the incorporation

of its phosphorylated derivative into DNA. The metabolic

conversion of SF to corresponding 59-monophosphate derivative

is more complex than that of other thiopurines [2].

Since SF utilizes different metabolic pathways for its intracel-

lular activation, SF treatment does not induce resistance in cancer

cells. In contrast, the deletion of a single enzyme responsible for

the metabolic activation of other purine nucleoside analogs is

enough for the development of resistance. SF better penetrates the

central nervous system (CNS) than its parental molecule - 6-

thioguanosine and is more effective in cancer treatment. SF is

useful against malignancies resistant to other thiopurines [3].

Despite limitations for their use, some purine analogs closely

related to SF showed considerable anti-angiogenic activities that

deserve scientific attention [4].

The metabolism of SF involves the cells’ glutathione system. SF

readily adducts to sulfhydryl compounds (glutathione and cysteine)

and by suppressing the glutathione detoxification system and

elevating the concentration of reactive oxygen species (ROS), SF

may induce the death of cancer cells [2].

In view of its considerable efficacy in cancer treatment and

moderate toxicity to normal cells [2], SF is suitable for combining

with other chemotherapeutic agents. SF acts synergistically with

doxorubicin (DOX), curcumine (CUR) and verapamil (VER) in

non-small cell lung cancer (NSCLC) cell lines [5–7]. The

effectiveness of the combined application with SF allowed the

use of these drugs at lower concentrations that are less toxic with

fewer adverse effects. We hypothesized that all mentioned anti-

cancer effects of SF could be useful for the reversion of resistance

to chemotherapeutics.

PLOS ONE | www.plosone.org 1 January 2013 | Volume 8 | Issue 1 | e54044

Multi-drug resistance (MDR) is the main limitation for the

accomplishment of successful cancer treatment. MDR phenotype

often relates to the over-expression of P-glycoprotein (P-gp), a

membrane transporter that effectively extrudes the cytotoxic drugs

from cancer cells and changes their pharmacokinetics. P-gp acts as

an efflux pump for various hydrophobic anticancer drugs such as

anthracyclines, vinca alkaloids, taxanes, epipodophyllotoxins, and

some of the new drugs (e.g. imatinib, nilotinib, everolimus). P-gp

over-expression is common in experimental cancer models as well

as in cancerous tissues from patients [8]. Therefore, P-gp has

become a main therapeutic target for overcoming MDR.

Among many options for reverting MDR, the agents with an

anti-cancer activity of their own could be examined as potential

MDR modulators. We speculated earlier that besides the synergy

between SF and DOX as anti-cancer drugs acting through

separate pathways, the alterations of MDR-related genes expres-

sion and reduction of P-gp activity could contribute to the chemo-

sensitization effect of SF [5], [6].

Therefore, we conducted further investigation of mechanisms

involved in SF action in resistant and incurable cancers. For that

purpose, we employed two different MDR cancer cell lines with

the over-expression of P-gp (NCI-H460/R and U87-TxR) [9],

[10]. We studied the potential of SF to kill resistant cancer cells

and induce autophagy as well as to modulate the mechanisms

involved in cancer progression, such as glutathione (GSH)

detoxification system, P-gp mediated drug transport, vascular

endothelial growth factor (VEGF) expression and.secretion. We

found that the modification of redox status by SF led to the

decrease in the expression of hypoxia inducible factor-1a (HIF-1a)

which regulates the expression of P-gp and VEGF. Thus, the

modulation of MDR by SF is the consequence of GSH

detoxification system suppression.

Materials and Methods

DrugsSF ([R,S]-2-amino-9-b-D-ribofuranosylpurine-6-sulfinamide)

was synthesized from 6-thioguanosine according to the published

procedure [1]. DOX solution was obtained from EBEWE

Arzneimittel GmbH, Vienna, Austria. R6Verapamil (Dex-VER)

was purchased from Sigma-Aldrich Chemie GmbH, Germany.

Tariquidar (TQ) was kindly provided by Dr. Sven Rottenberg

from The Netherlands Cancer Institute, Amsterdam. CoCl2 was

obtained from Fisher Scientific, USA. SF was kept at 220uC.

Before treatment, SF and CoCl2 were freshly diluted in water,

while aliquots of DOX were thawed from 220uC. Dex-VER was

kept as 1 mM stock solution at room temperature. TQ was diluted

in dimethyl sulfoxide (DMSO) and 10 mM aliquots were kept at

220uC.

ChemicalsRPMI 1640 medium, Minimum Essential Medium (MEM),

penicillin-streptomycin solution, antibiotic-antimycotic solution,

L-glutamine and trypsin/EDTA were purchased from PAA,

Vienna, Austria. Fetal bovine serum (FBS), sulforhodamine B

(SRB) and acridine orange were obtained from Sigma-Aldrich

Chemie GmbH, Germany. Matrigel was kindly provided by Dr.

Sanja Mijatovic from the Institute for Biological Research ‘‘Sinisa

Stankovic’’, University of Belgrade, Serbia. Propidium iodide (PI)

was purchased from Roche Applied Science, Basel, Switzerland

and Annexin-V-FITC (AV) from Abcam, Cambridge, UK. FITC-

conjugated anti-P-gp antibody was provided by BD Biosciences,

United Kingdom, while PE-conjugated anti-VEGF antibody was

obtained from R&D Systems, Minneapolis, MN USA. Carboxy-

fluorescein succinimidyl ester (CFSE), dihydroethidium (DHE) was

obtained from Molecular ProbesH, Invitrogen, CA, USA. Primary

antibodies against caspase 3 and b-actin were purchased from Cell

Signaling Technology Inc, Danvers, MA, USA, while primary

antibody against gamma-glutamylcysteine synthetase (cGCS) was

a kind gift from Dr Bato Korac, Institute for Biological Research

‘‘Sinisa Stankovic’’, University of Belgrade, Serbia. Peroxidase-

conjugated goat anti-rabbit IgG was obtained from Jackson

ImmunoResearch Laboratories Inc, West Grove, PA, USA.

Cells and Cell CultureNCI-H460 and U87 cell lines were purchased from the

American Type Culture Collection, Rockville, MD. NCI-H460

cells were maintained in RPMI 1640 supplemented with 10%

FBS, 2 mM L-glutamine, 4.5 g/L glucose, 10,000 U/mL penicil-

lin, 10 mg/mL streptomycin, 25 mg/mL amphotericin B solution

at 37uC in a humidified 5% CO2 atmosphere. NCI-H460/R cells

were originally selected from NCI-H460 cells in our laboratory

and cultured in a medium containing 100 nM DOX as described

previously [9]. U87 cells were cultured in MEM supplemented

with 10% FBS, L-glutamine (2 mM) and 5000 U/ml penicilin,

5 mg/ml streptomycin solution. U87-TxR cells were selected from

U87 cells in our laboratory after continuous exposure to stepwise

increasing concentrations of paclitaxel (100–300 nM) for a period

of 9 months as already published [10]. HaCaT cell line (normal

human keratinocytes obtained from CLS - Cell Lines Service,

Eppelheim, Germany) was generous gift from Prof. Andra Jorg,

Division of Biophysics, Research Center Borstel, Leibniz-Center

for Medicine and Biosciences, Borstel, Germany. HaCaT cells

were cultured in DMEM supplemented with 10% FBS, 4 g/L

glucose, L-glutamine (2 mM) and 5000 U/ml penicilin, 5 mg/ml

streptomycin solution. MDR cancer cells were sub-cultured at

72 h intervals using 0.25% trypsin/EDTA and seeded into a fresh

medium at the following densities: 8,000 cells/cm2 for NCI-H460,

16,000 cells/cm2 for NCI-H460/R and U87, 32,000 cells/cm2

for U87-TxR. HaCaT cells were sub-cultured at 144 h intervals

using 0.25% trypsin/EDTA and seeded into a fresh medium at 64,

000 cells/cm2.

Sulforhodamine B AssayCells grown in 25 cm2 tissue flasks were trypsinized, seeded into

flat-bottomed 96-well tissue culture plates and incubated over-

Figure 1. Chemical structure of purine nucleoside analog –sulfinosine (SF).doi:10.1371/journal.pone.0054044.g001

Sulfinosine Modulates Cancer Progression


night. Investigated cell lines NCI-H460, NCI-H460/R, U87, U87-

TxR and HaCaT cells were seeded at 4, 000, 8,000, 8,000 and

16,000 cells/well, respectively. SF treatment (1–100 mM) lasted

72 h. The cellular proteins were stained with sulforhodamine B

(SRB) assay, following slightly modified protocol of Skehan et al

[11]. Briefly, the cells in 96-well plates were fixed in 50%

trichloroacetic acid (50 mL/well) for 1 h at 4uC, rinsed in tap

water and stained with 0.4% (w/v) sulforhodamine B in 1% acetic

acid (50 mL/well) for 30 min at room temperature. The cells were

then rinsed three times in 1% acetic acid to remove the unbound

stain. The protein-bound stain was extracted with 200 mL 10 mM

Tris base (pH 10.5) per well. The optical density was read at

540 nm, with correction at 670 nm (LKB 5060-006 Micro Plate

Reader, Vienna, Austria).

Matrigel GrowthFor three-dimensional (3-D) cultures, cells were plated at the

same densities as for two (2-D) cultures onto reconstituted (pre-

gelled) basement membrane (Matrigel; BD Biosciences, San Jose,

CA, USA) in RPMI 1640 medium with 10% FBS. Cells were

incubated for 72 h and photographed live by phase microscopy.

Determination of Cell Proliferation (CFSE Staining)The rate of cell proliferation was measured by flow-cytometric

analysis of cells labeled with carboxyfluorescein succinimidyl ester

or CFSE [12]. Briefly, detached cells (56106 cells/mL) were

stained with 1 mM CFSE for 10 min in dark at 37uC, washed

twice in fresh medium, seeded in six-well plates at 56104 per well,

and then exposed to SF. After 72 h of cultivation, cells were

trypsinized and washed twice in PBS. Finally, the cells were

resuspended in PBS and analyzed by flow-cytometry. Green

fluorescence emission was measured using a FACSCalibur flow-

cytometer (Becton Dickinson, Oxford, United Kingdom) and

analyzed using Cell-Quest software.

Cell Death DetectionThe percentages of apoptotic, necrotic and viable cells were

determined by Annexin-V-FITC (AV) and propidium iodide (PI)

labeling. NCI-H460/R and U87-TxR cells were plated and

incubated overnight in 6-well plates at density of 80,000 and

160,000 cells/well, respectively. After 72 h of SF treatment, the

attached and floating cells were collected by centrifugation. The

cells pellet was re-suspended in 100 mL of binding buffer

containing 10 mM HEPES/NaOH, 140 mM NaCl, 5 mM CaCl2(pH 7.4), supplemented with 0.2 mg AV and 1 mg PI. After the

incubation period (30 min at 37uC in dark), additional 400 mL of

binding buffer was added and AV/PI staining was analyzed within

1 h by flow-cytometry. The fluorescence intensity (green FL1-H

and red FL2-H) was measured on FACSClibur flow-cytometer

(Becton Dickinson, Oxford, United Kingdom). In each sample,

10,000 cells were recorded (gated to exclude cell debris), and the

percentages of viable (AV2 PI2), early apoptotic (AV+ PI2),

apoptotic and necrotic (AV+ PI+), and already dead (AV2 PI+)

cells were analyzed by CellQuest Pro data analysis software.

Caspase ActivationActivation of caspases was measured by flow-cytometry after

labelling the cells with a cell-permeable, FITC-conjugated pan-

caspases inhibitor (ApoStat; R&D Systems, Minneapolis, MN)

according to the manufacturers instructions. The increase in green

fluorescence (FL1-H) as a measure of caspase activity within

individual cells of the treated population was determined using

FACSCalibur flow-cytometer (Becton Dickinson, Oxford, United

Kingdom).

Autophagy AssessmentThe appearance of acidic autophagic vesicles was detected by

flow-cytometry. After SF treatment cells were trypsinized, washed

and incubated for 15 min at 37uC with 1 mM acridine orange.

Acridine orange-stained cell nuclei are fluorescent green, while

autophagic lysosomes are fluorescent orange-red. The increase in

red vs. green (FL3/FL1) fluorescence ratio, reflecting the

autophagy, was determined using a FACSCalibur flow-cytometer

(Becton Dickinson, Oxford, United Kingdom) and Cell Quest Pro

software.

Western BlotCells grown in 100 mm Petri dishes at following densities:

400,000 cells per dish for NCI-H460/R and 750,000 per dish for

U87-TxR were lysed after SF treatment with lysis buffer (30 mM

Tris-HCl pH 8.9, 150 mM NaCl, 1% NP-40) containing 1 mM

phenylmethylsulfonyl fluoride and protease inhibitor cocktail

(Sigma-Aldrich Chemie GmbH, Germany). After 30 min on ice,

samples were centrifuged at 14 000 g for 15 min at 4uC, and

supernatants were collected. Equal amounts of protein from each

sample was separated by SDS-PAGE on 6–15% gels and

transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA,

USA). Following incubation with primary antibodies against

caspase 3, b-actin and gamma glutamylcstein synthetase (cGCS)

and peroxidase-conjugated goat anti-rabbit IgG as the secondary

antibody, specific protein bands were visualized using Amersham

ECL reagent (GE Healthcare Life Sciences, UK). The protein

levels of cGCS were quantified by densitometry using ImageJ

software and expressed relative to b-actin.

DHE StainingFlow-cytometric measurements of dihydroethidium (DHE)-

fluorescence were used to measure ROS concentration in MDR

cancer cells. Adherent cells were rinsed with PBS and harvested by

trypsinization. Cells were incubated in PBS with 10% FBS and

10 mM DHE for 45 min. DHE-fluorescence was analyzed by flow-

cytometry (excitation 488 nm, and emission 585 nm, FL2-H

channel). Mean fluorescence intensity (MFI) was calculated after

correction for autofluorescence.

Colorimetric Detection of Glutathione (GSH)Cells grown in 25 cm2 tissue flasks were trypsinized and

counted. The same number of cells (2.56106) for each sample

was exposed to further procedure. Briefly, the cells were collected

by centrifugation at 7006g for 5 minutes at 4uC and the

supernatant was removed. Then, the cell pellet was resuspended in

0.5 ml ice-cold PBS and centrifuged at 7006g for another 5

Figure 2. SF inhibits the cell growth and suppresses the cell proliferation. The growth inhibitory effects of SF on NCI-H460 and NCI-H460/R(A), U87, U87-TxR and HaCaT (C) cells grown on plastic after 72 h treatment were assessed by SRB assay. Average 6 S.D. values were calculated fromfive independent experiments (n = 5). NCI-H460 and NCI-H460/R (B), U87 and U87-TxR (D) cells were stained with CFSE and incubated for 72 h with10 mM SF. The rate of proliferation (CFSE declination) was determined by flow-cytometry on channel FL1. Light microscopy of NCI-H460 and NCI-H460/R (E), U87 and U87-TxR (F) cell growth on plastic (2-D culture) and matrigel growth (3-D culture) after 72 h of 10 mM SF treatment.doi:10.1371/journal.pone.0054044.g002



Figure 3. SF induces caspase activation, apoptosis and autophagy in MDR cancer cell lines. Cell death analysis of NCI-H460/R (A) andU87-TxR (C) cells untreated and treated with 5 mM SF (B, D) for 72 h. The samples were analyzed for green fuorescence (Annexin-V-FITC) and redfluorescence (Propidium Iodide) by flow-cytometry. The assay distinguishes viable cells (AV2 PI2), apoptotic cells (AV+ PI2), late apoptotic andnecrotic cells (AV+ PI+) and secondary necrotic or dead cells (AV2 PI+). Pancaspase activation in NCI-H460/R (E) and U87-TxR (F) cells untreated andtreated with 10 mM SF was assessed after 72 h by flow-cytometry. The cells were labelled with a cell-permeable, FITC-conjugated pan-caspasesinhibitor (ApoStat). Western blot analysis of procaspase-3, p17 cleaved caspase-3 and b-actin in NCI-H460/R (G) and U87-TxR (H) cells. (I) Theautophagy in NCI-H460/R and U87-TxR cell lines assessed after 144 h incubation period (72 h of SF treatment followed by next 72 h of cell recovery infresh medium). Fold of increase in red vs. green (FL3/FL1) fluorescence ratio after acridine orange-staining was determined using a FacsCalibur flowcytometer and Cell Quest Pro software. The statistical significance between the controls and treatments is presented as p,0.01 (**) and p,0.001(***).doi:10.1371/journal.pone.0054044.g003



minutes at 4uC. The supernatant was removed and the cells were

lysed in 80 ml ice-cold Glutathione Buffer (GSH Colorimetric

Detection Kit, Bio-Vision, CA) for 10 minutes on ice. Then, 20 ml

of 5% Sulfosalicylic Acid was added and the samples were

centrifuged at 80006g for 10 minutes at 4uC. The supernatant was

transferred to a fresh tube and used for GSH assay. Glutathione

Buffer was added to each well (96-well plate) at a volume of 160 ml

and incubated 10 minutes at room temperature. Afterwards, 20 ml

of either prepared standards or samples was added to each well

and incubated for another 10 minutes at room temperature.

Finally, 20 ml of Substrate Solution (GSH Colorimetric Detection

Kit, BioVision, CA) was added and the absorbance of generated

product (2-nitro-5-thiobenzoic acid) was read at 405 nm (LKB

5060–006 Micro Plate Reader, Vienna, Austria). The concentra-

tions of GSH were determined by using the standard GSH

calibration curve and related to the concentrations of proteins in

cell lysates. The GSH detection for each sample was performed at

least six times.

RNA Extraction and RT-PCRTotal RNA was extracted from untreated NCI-H460/R and

U87-TxR cells and the cells treated with SF. The isolation was

carried out using Trizol (Invitrogen Life Technologies, CA, USA)

according to the manufacturer’s instructions. RNA was quantified

on spectrophotometer and quality was determined by agarose gel

electrophoresis. Reverse transcription (RT) reactions using 25 mg

total RNA were performed with oligo-dT primers using M-MLV

Reverse Transcriptase (Gibco BRL, USA) following the manufac-

turer’s instructions. PCR reactions were performed with primers

specific for, gst-p, vegf, mdr1 and hif-1a [13–16], b-actin [17] and

gapdh [18] was used as an internal control and co-amplified with

genes of interest in all PCR experiments.

The PCR reactions were performed on the GeneAmpH PCR

System 9700 (Applied Biosystems, CA, USA) under the following

conditions for hif-1a, mdr1 and gst-p: initial denaturation at 95uCfor 5 min, 24, 25 or 28 cycles (respectively) at 95uC for 15 s, 56uCfor 30 s, 72uC for 30 s and at 4uC indefinitely. When PCR was

performed to determine the expression of the vegf gene, 35 cycles

were applied with the annealing temperature of 62uC. The gapdh

primers were used at following ratios: 1:4 to the mdr1 primers and

Figure 4. SF increases ROS concentration and inhibits GSH detoxification in MDR cancer cell lines. Flow-cytometric measurements ofdihydroethidium (DHE)-fluorescence correspond to cellular ROS levels (A). Mean fluorescence intensity (MFI) calculated after correction forautofluorescence is presented. The data indicate the average 6 S.D. of three experiments carried out in triplicate. Statistical significance of SFtreatment compared to untreated control: p,0.01 (**) and p,0.001 (***). Detection of GSH concentration (B) for each sample was performed at leastsix times. The data indicate the average 6 S.D. Statistical significance of SF treatment compared to untreated control: p,0.01 (**). The expression ofgst-p mRNA in NCI-H460/R (C) and U87-TxR cells (D) was evaluated relative to the internal control - b-actin. The PCR products were separated onagarose gels beside a 100 bp DNA ladder. Statistical significance between treated and untreated cells is presented as p,0.001 (***).The decreases incGCS expression in NCI-H460/R (E) and U87-TxR cells (F) after SF treatments were calculated relative to b-actin expression and untreated samples.Statistical significance between treated and untreated cells is presented as p,0.01 (**).doi:10.1371/journal.pone.0054044.g004

Figure 5. SF decreases the expression of mRNAs involved in tumor progression. The expression of gst-p, hif-1a, mdr1 and vegf mRNAs inNCI-H460/R (A) and U87-TxR cells (B) after 10 mM SF treatment was evaluated relative to the internal control – gapdh and b-actin, and calculated inrelation to the untreated control. Statistical significance between treated and untreated cells is presented as p,0.05 (*) and p,0.01 (**). Relative hif-1a (C), mdr1 (D) and vegf (E) mRNAs expression after 10 mM SF treatment in normoxic (absence of CoCl2) and hypoxic (presence of 50 mM CoCl2)conditions studied in NCI-H460/R cells. Statistical significance between SF treated and SF untreated cells is presented as p,0.05 (*) and p,0.01(**).Statistical significance between CoCl2 treated and CoCl2 untreated cells is presented as p,0.01 (##) and p,0.001 (###).doi:10.1371/journal.pone.0054044.g005



Figure 6. SF inhibits P-gp expression and increases DOX accumulation. P-gp expression was detected after 24 h, 48 h and 72 h in NCI-H460/R (A) and U87-TxR (B) cells untreated and treated with 5 and 10 mM SF. The percentage of P-gp positive cells (stained with FITC-labeled directantibody) was determined after elimination of the cells that were stained with FITC-labeled isotypic antibody. The corresponding flow-cytometrichistograms illustrate the effect of 10 mM SF after 72 h in NCI-H460/R (C) and U87-TxR (D) cells. DOX accumulation in NCI-H460/R (E) and U87-TxR (F)cells untreated and pretreated with, 5 and 10 mM SF. DOX accumulation was assessed at each time point (24 h, 48 h and 72 h) after 120 min of 20 mMDOX treatment. Flow cytometric profiles after 72 h pretratment compare the effects of 10 mM SF, 50 nM TQ and 10 mM Dex-VER in NCI-H460/R (G)and U87-TxR (H) cells Three independent experiments were performed (a minimum of 10,000 events were collected for each experimental sample).Statistical significance between treated and untreated cells is presented as p,0.05 (*), p,0.01 (**) and p,0.001 (***).doi:10.1371/journal.pone.0054044.g006



1:6 to the hif-1a primers in order to attain linear amplification

conditions. The b-actin primers were used at following ratios: 1:2 to

the gst-p primers and 1:5 to the vegf primers in order to attain linear

amplification conditions. The PCR products were separated in 2%

agarose gels stained with ethidium bromide. Multi-Analyst/PC

Software Image Analysis (Bio-Rad Gel Doc 1000, CA, USA) was

employed for densitometry analysis.

DOX Accumulation AssayDOX accumulation was analyzed by flow-cytometry utilizing

the ability of DOX to emit fluorescence. The intensity of the

fluorescence was proportional to DOX accumulation. Studies

were carried out after 24 h, 48 h and 72 h SF treatment. NCI-

H460/R and U87-TxR cells were cultured in 25 cm2 flasks,

trypsinized and re-suspended in 10 mL centrifuge tubes in a

DOX-containing medium (20 mM). Then, the cells were incubat-

ed at 37uC in 5% CO2 for 120 min. At the end of the

accumulation period, the cells were pelleted by centrifugation,

washed with phosphate buffered saline (PBS) and placed in cold

PBS. The samples were kept on ice in dark until the analysis on

FACScalibur flow-cytometer (Becton Dickinson, Oxford, United

Kingdom). The fluorescence of DOX was assessed on fluorescence

channel 2 (FL2-H) at 530 nm. A minimum of 10,000 events were

assayed for each sample. The differences in curve shape were

quantified using a Komogorov-Smirnov nonparametric statistic. P

values were calculated (available on request) in CellQuest Pro and

run on a Macintosh computer.

Flow-cytometric Analysis of P-gp and VEGF ExpressionFlow-cytometry was used to measure P-gp and VEGF

expression levels in MDR cancer cells. Untreated and SF treated

cells (26105) were collected by trypsinization, washed in ice-cold

PBS, and then directly immuno-stained by FITC-conjugated anti-

P-gp antibody according to the manufacturers’ protocol (BD

Biosciences, United Kingdom). An isotype control IgG2bk(Abcam, Cambridge, United Kingdom) was evaluated for each

experimental sample to discriminate the level of background

fluorescence of negative cells. For VEGF expression analysis, the

cells were fixed in 4% paraformaldehyde, 10 min at room

temperature, washed and resuspended at saponin 0.05% (w/v)

buffer and incubated with PE-conjugated anti-VEGF antibody

according to the manufacturers’ protocol (R&D Systems, USA).

An isotype control IgG2a (Abcam, Cambridge, United Kingdom)

was evaluated for each experimental sample to discriminate the

level of background fluorescence of negative cells. Mean fluores-

cence intensity was determined for positively stained cells. The

samples were kept on ice in dark until the analysis on FACScalibur

flow-cytometer (Becton Dickinson, Oxford, United Kingdom).

The fluorescence of FITC-conjugated anti-P-gp was assessed on

fluorescence channel 1 (FL1-H) at 530 nm, while PE-conjugated

anti-VEGF was assessed on fluorescence channel 2 (FL2-H) at

585 nm. A minimum of 10,000 events were assayed for each

sample (the gate excluded cell debris and dead cells) and the

obtained results were analysed using Cell Quest Pro Software

(Becton Dickinson, Oxford, United Kingdom).

MTT AssayCell metabolic activity was assessed by the MTT assay based on

the reduction of 3-(4,5-dimethyl-2-thizolyl)-2,5-diphenyl-2H-tetra-

zolium bromide (MTT, Sigma, St Louis, MO) into formazan dye

by active mitochondria of living cells. The combined effects of

simultaneous and subsequent treatment were studied on MDR

cancer cell lines. NCI-H460/R and U87-TxR cells prepared for

simultaneous treatment were seeded at 4, 000 and 8,000 cells/

well, respectively. SF treatment (5 mM) in combination with

different DOX concentrations lasted 72 h. The subsequent

treatments were performed on NCI-H460/R and U87-TxR cells

initially seeded at lower densities (500 cells/well and 1,000 cells/

well, respectively). Pretreatment with 5 mM SF lasted for 72 h and

was followed by additional 72 h treatment with different

concentrations of DOX. MTT was added to final concentration

of 0.1 mg/ml in each well of a 96-well microplate and plates were

incubated at 37uC for 4 h. Then, DMSO was added to dissolve

formazan product, which amount was proportional to the number

of live cells. The absorbance of dissolved dye was measured at

540 nm using an automatic microplate reader (LKB 5060-006

Micro Plate Reader, Vienna, Austria). Growth inhibition (I) was

determined according to the following equitation:

I %ð Þ~ 1� A treated sample=A untreated controlð Þð Þ| 100

where A is for absorbance.

IC50 value was defined as concentration of each drug that

inhibited cell growth by 50%. IC50 was calculated by linear

regression analysis using Excel software.

ELISA for Detection of Human VEGF165 in Cell CultureMedium

MDR cells (NCI-H460/R and U87-TxR), seeded in 6-well

plates, were incubated overnight and then treated with SF. The

cell medium (supernatant) was collected 24 h, 48 h and 72 h after

treatment for determination of secreted VEGF165 protein by

VEGF immunoassay kit (Quantikine Human VEGF ELISA Kit,

R&D Systems, Minneapolis, USA). The procedure was complied

according to the manufacturers’ manual. The results were

normalized based on the same amount of cells analyzed. A

standard curve was generated using recombinant VEGF165

supplied with the kit. The concentrations of VEGF in cell-free

culture supernatants were examined in triplicates in two indepen-

dent experiments.

Table 1. Relative reversion of resistance to DOX insimultaneous and subsequent treatments with SF.

Cell Lines Drugs IC50 (mM)RelativeReversion

NCI-H460/R DOX 3.25060.260

SF 5 mM (+ DOX)

simultaneously 3.03660.091 1.07

DOX 7.08360.212

SF 5 mM (+ DOX)

subsequently 0.97660.010 7.26

U87-TxR DOX 0.22460.027

SF 5 mM (+ DOX)

simultaneously 0.09860.025 2.28

DOX 4.10060.079

SF 5 mM (+ DOX)

subsequently 0.24160.019 17.01

doi:10.1371/journal.pone.0054044.t001



Statistical AnalysisStatistical analysis was performed by Statistica 6.0 software. The

results were tested for normality. If obtained values were not

normally distributed, the groups were compared by Student’s t –

test. For normally distributed variables, one-way analysis of

variance (ANOVA) was used. When statistical significance was

observed, the Tukey honest significant difference (HSD) test was

applied. Statistical significance was accepted if p,0.05 (*), p,0.01

(**), p,0.001 (***).

Results and Discussion

SF Inhibits the Growth of MDR Cancer CellsWe established NSCLC and glioblastoma P-gp over-expressing

cell lines (NCI-H460/R and U87-TxR) with MDR phenotype in

order to investigate potential anti-cancer agents [9], [10]. NCI-

H460/R and U87-TxR are MDR cancer cell lines that originated

from NCI-H460 (NSCLC cell line) and U87 (glioblastoma cell

line). The parental cell lines were considered as sensitive since the

cells derived from patients who had not undergone chemotherapy.

In the present study, we tried to elucidate the action of sulfinosine

(SF), a synthetic purine nucleoside analog, in these two MDR

cancer cell lines. We choose SF because of evidences that its

therapeutically effective concentrations could not induce the

resistance. SF also efficiently penetrates to CNS [2]. Moreover,

recent clinical study showed that the combination therapy

including 6-thioguanine (closely related molecule to SF) is

promising for patients with recurrent high-grade anaplastic glioma

[19].

The effects of SF on cancer cell growth after 72 h treatment

were evaluated by the chemo-sensitivity assay - sulforhodamine B

(SRB). SF inhibited the growth of sensitive and MDR cancer cell

lines in a dose-dependent manner (Figure 2A, C). Since

application of anti-cancer agents is limited by their toxicity

towards normal cells, we tested the effect of SF on HaCaT cells

(normal human keratinocytes). The effects on growth of HaCaT

after continuous treatment of 72 h were assessed also by SRB

assay. SF did not reduce significantly the number of normal cells

even with the highest concentration (100 mM) (Figure 2C). We

demonstrated that SF inhibits the growth of sensitive as well as

resistant NSCLC and glioblastoma cells in micro-molar range of

concentrations, and that its efficacy was not affected by the

presence of the MDR phenotype. Moreover, SF was non-toxic to

normal cells (HaCaT) in the range of concentrations necessary to

inhibit the growth of cancer cells.

Next, we evaluated the cytostatic effect of 10 mM SF in each cell

line by CFSE staining (Figure 2B, D). CFSE is a vital dye stable in

the cytoplasm for about 7–8 generations, but the intensity of CFSE

fluorescence declines due to its progressive halving within daughter

cells following each cell division. In that way, the CFSE

distribution in the cells can estimate the rate of cell proliferation.

The CFSE distribution in control and SF treated samples is

illustrated by flow-cytometric profiled histograms (Figure 2B, D).

The inhibition of proliferation observed in the presence of SF was

the most pronunced in NCI-H460/R cells. These results indicated

that the inhibition of proliferation is partly responsible for the anti-

cancer activity of SF.

Since NSCLC and glioblastoma cell lines have high metastatic

potential, we compared the effects of SF to inhibit the cell growth

after 72 h on plastic (2-D culture) and in reconstituted basement

membrane – matrigel (3-D culture) (Figure 2E, F). We found that

SF inhibited the growth in 3-D culture with the same efficacy

observed in 2-D culture. The fact that the cells were detached

from each other after SF treatment in matrigel, especially

glioblastoma cells, points to the change in their adhesive

properties. Therefore, we speculate that SF could affect the

affinity of cancer cells to invade the blood vessels, induce tumor-

angiogenesis and metastasis.

SF Induces Caspase-dependent Apoptosis in MDR CancerCells

Next, we proceeded to examine whether induction of apoptosis

contributes to the anti-cancer action of SF in MDR cancer cell

lines. To assess the apoptosis induced by SF after 72 h the cells

were seeded at optimal density for their growing properties. In that

way, the untreated controls did not achieve confluence at the end

of incubation period. Annexin-V-FITC/Propidium Iodide stain-

ing revealed that SF increases the proportion of apoptotic cells

(AV+PI2) in both MDR cancer cell lines. The results are

summarized in (Figure 3A, B, C, D): alive cells are negative for

both, Annexin-V and Propidium Iodide (AV2PI2); apoptotic

cells only bind Annexin-V (AV+ PI2), apoptotic and necrotic cells

are Annexin-V and Propidium Iodide positive (AV+ PI+) and

secondary necrotic cells, that were already dead, are positive only

for Propidium Iodide (AV2 PI+). The percentages of apoptotic

and necrotic cells were increased in samples treated with 5 mM SF

(Figure 3B, D).

Caspases play a key role in the apoptotic pathway. Initiator

caspases, which include caspases-2, -8, -9 and -10, activate the

caspase cascade through the removal of the inactive prodomains of

the effector caspases. Once activated, effector caspases, including

caspases-3, -6 and -7, cleave several dozen key substrates within

the cell in order to carry out the apoptotic process [20]. We

showed by a fluorochrome-labeled pan-caspase inhibitor ApoStat

that SF induced the activation of caspases (Figure 3E, F). SF

caused the shift of ApoStat flow-cytometric profile by 83.5% and

28.6% in NCI-H460/R and U87-TxR cells, respectively

(Figure 3E, F). We found that the induction of apoptosis by SF

after 72 h was caspase-dependent in both resistant cell lines.

However, the caspase activation was more pronounced in resistant

NSCLC than in glioblastoma cells. In order to find if an effector

caspase-3 was activated after SF treatment, we analyzed the

Figure 7. SF modulates VEGF165 expression and secretion in MDR cancer cell lines. The amplified NCI-H460/R (A) and U87-TxR samples (B)of the vegf gene (four splicing products of vegf mRNA) were visualized with ethidium-bromide in agarose gel next to a DNA ladder (100 bp). The PCRproduct of b-actin was co-amplified with vegf. The relative expression of vegf165 was calculated in relation to b-actin expression. Statisticalsignificance between treated and untreated cells is presented as p,0.05 (*) and p,0.001 (***). VEGF expression was detected after 24 h, 48 h and72 h in NCI-H460/R (C) and U87-TxR (D) cells untreated and treated with 10 mM SF. The mean fluorescence of VEGF (PE-labeled direct antibody) wasdetermined after elimination of the signal obtained with PE-labeled isotypic antibody. The corresponding flow-cytometric histograms illustrate theeffect of 10 mM SF after 24 h in NCI-H460/R (E) and 48 h in U87-TxR (F) cells. VEGF secretion levels in culture medium were evaluated after 24 h, 48 h,and 72 h using Human VEGF Immunoassay Kit. NCI-H460/R (G) and U87-TxR cells (H) were treated with 10 mM SF. The data indicate the average 6S.D. of four experiments carried out in triplicate. Statistical significance of SF treatment compared to untreated control when the secretion levels werenormalized based on the same amount of cells analyzed in untreated and treated samples, grey bars: p,0.05 (*) and p,0.01 (**). Statisticalsignificance of SF treatment compared to untreated control when the secretion levels of treated cells were not normalized, black bars: p,0.05 (*) andp,0.01 (**).doi:10.1371/journal.pone.0054044.g007



expression of its precursor procaspase-3 (35 kDa) and cleaved

caspase-3 (17 kDa) by Western blotting (Figure 3G, H). The results

revealed the obvious increase in 17 kDa form after 5 and 10 mM

SF application in both MDR cancer cell lines. Although, the

expression of procaspase-3 was not affected by SF treatment, the

increase of cleaved form corresponds to the activation of caspase-

3.

Our previous study revealed that p53 has undergone mutations

during induction of resistance in NSCLC (NCI-H460/R) cells [6].

p53 mutation is often associated with increased resistance to

chemotherapy [21]. However, the anti-cancer action of SF

certainty included induction of apoptosis in resistant NSCLC

cells. The proposed mechanism for SF anti-cancer action involves

the formation of adducts with sulfhydryl compounds - glutathione

and cysteine [22], [23]. Depletion of glutathione pool may cause

down-regulation of Bcl-2 [24], release of cytochrom c from

mitochondria and activation of caspases [25]. Therefore, the cell

death induced by SF in resistant NSCLC cell line could be p53-

independent.

Several anti-apoptotic mechanisms, such as over-expression of

PKA, HSP70, Bcl-2 and deficiency in PTEN are responsible for

the evading apoptosis in glioblastoma sensitive cell line (U87) [26–

29]. We assume that the same mechanisms are present in resistant

glioblastoma cell line (U87-TxR). However, U87-TxR cells were

susceptible to the induction of cell death by SF.

Autophagy, a catabolic process responsible for the removal of

long-lived proteins and damaged organelles through the lysosomal

machinery, and apoptosis may be induced by common upstream

signals, and thus results in combined autophagy and apoptosis.

Under certain circumstances, autophagy constitutes a stress

adaptation that avoids cell death and suppresses apoptosis [30].

In order to reveal the possible connection between SF driven

apoptosis and autophagy, we investigated by flow-cytometry

whether SF could induce the appearance of autophagy-associated

acidic vesicles (Figure 3I). The changes in the level of autophagy

were obtained after removal of SF from medium. Significant

increases in autophagy were observed after 5 and 10 mM SF

treatments in NCI-H460/R and U87-TxR cells, respectively

(Figure 3I).

SF Modulates Detoxification Capacity of MDR CancerCells

Cellular redox environment is a delicate balance between the

levels of reactive oxygen species (ROS i.e., superoxide and

hydrogen peroxide) and the antioxidant system that scavenges

them (i.e., glutathione/glutathione peroxidase and tioredoxin/

peroxiredoxin pathways) [31]. Considering that ROS can be

generated from exogenous sources (e.g. ionizing radiation,

chemicals), we examined whether SF changes the ROS concen-

tration in MDR cancer cells. We used dihydroethidium (DHE)-

fluorescence and flow-cytometry to measure the ROS levels. DHE

is a non-fluorescent chemical that upon entry into the cell

undergoes oxidation to yield the fluorescent chemical, 2-hydro-

xyethidium (HE). HE intercalates with DNA and shows strong

fluorescence in presence of superoxide radical [32]. Indeed,

10 mM SF significantly increased the ROS concentration in both

resistant cancer cell lines, while even 5 mM SF induced consid-

erable change in NCI-H460/R cells (Figure 4A).

The main cellular antioxidant detoxification pathway – GSH

system scavenges ROS and prevents them from causing intracel-

lular damage including lipid peroxidation, DNA damage and

protein oxidation [33]. Therefore, we studied the effects of SF on

GSH concentration. The concentration of reduced glutathione

(GSH) was measured in untreated cells and cells treated for 72 h

with 5 and 10 mM SF. SF caused strong and significant depletion

of GSH in both resistant cancer cell lines (Figure 4B). After 10 mM

SF treatments, the concentrations decreased from19 to 12 mg

GSH/ml protein in NCI-H460/R and from 17 to 7 mg GSH/ml

protein in U87-TxR cells (Figure 4B).

We also measured the level of mRNA expression of the

common drug resistance marker glutathione-S-transferase p (gst-

p), which is a component of GSH system. Application of SF to the

resistant cell lines induced a significant reduction in gst-pexpression compared to untreated cells (Figure 4C, D). Herein,

we also showed that 10 mM SF significantly decreased the

expression of gamma Glutamyl-cysteine-synthetase (cGCS), the

enzyme critical for GSH synthesis (Figure 4E, F).

The role of GSH and related enzymes in cellular resistance to

xenobiotics, including chemotherapy, is well established. Clearly,

SF has a potential to modulate GSH system to therapeutic

advantage. SF significantly decreased intracellular GSH levels by

inhibition of its synthesis. This contributes further to the increase

of ROS, the induction of cell damage and eventually the cell

death.

SF Decreases the Expression of hif-1a and hif-1aRegulated mRNAs

We assumed that SF action through modification of redox status

(GSH depletion and ROS generation) could influence the

expression level of hypoxia inducible factor-1a (HIF-1a) and

indirectly decrease the expression of P-gp and VEGF. We found

that 10 mM SF significantly decreased the mRNA battery (gst-p,

hif-1a, mdr1 and vegf) in both MDR cancer cell lines (Figure 5A, B).

In order to reveal whether SF could regulate the HIF-1aexpression induced by hypoxia, MDR cancer cells were treated

with cobalt chloride (CoCl2) that stabilizes HIF-1a and induces

HIF-1 responsive genes similarly to that of hypoxic condition [16].

We showed that SF retained the potential to inhibit the expression

of hif-1a, mdr1 and vegf mRNAs even in hypoxic mimic condition in

NCI-H460/R cell line (Figure 5C, D, E).

SF Inhibits P-gp Expression and ActivityConsidering the obtained results on mRNAs expression,

especially mdr1 gene, next, we studied the potential of SF to

modulate MDR. Therefore, we assessed the P-gp (coded by mdr1)

expression by flow-cytometry (Figure 6A, B, C, D). We used direct

FITC-labeled antibody for P-gp to study the changes in protein

expression level. After determination of background fluorescence

with isotypic control antibody, we were able to define the portion

of P-gp positive cells in each tested sample. Significant decreases in

amount of P-gp positive NCI-H460/R and U87-TxR cells were

observed after SF treatment (Figure 6A, B). The effect of SF on P-

gp expression was concentration- and time-dependent. The best

effect was achieved after 72 h and illustrated by flow-cytometric

profiled histogram (Figure 6C, D). The fluorescence in SF treated

samples declined in comparison to untreated controls of NCI-

H460/R and U87-TxR by 34.1% and 36.9%, respectively. SF

clearly decreased the fraction of P-gp expressing cells in both

resistant cancer cell lines.

In order to investigate the effect of SF on P-gp function in NCI-

H460/R and U87-TxR cell lines, we analyzed intracellular

accumulation of chemotherapeutic (DOX), which is P-gp substrate

(Figure 6E, F, G, H). We confirmed that MDR in NCI-H460/R

and U87-TxR cell lines correlates with over-expression of P-gp

membrane transporter. Low intracellular accumulation of P-gp

substrates – DOX and Rhodamine 123 shown in these cell lines

was the consequence of high P-gp activity [9], [10].



The accumulation of DOX was assessed by flow-cytometry and

compared among SF, TQ (non-competitive P-gp inhibitor) and

Dex-VER (competitive P-gp inhibitor). Marked increases in DOX

accumulation were observed in NCI-H460/R and U87-TxR cells

pretreated with SF during 48 h and 72 h (Figure 6E, F). SF was

the most efficient in the modulation of P-gp activity after 72 h

showing the same potential as TQ and Dex-VER in both MDR

cancer cell lines (Figure 6G, H). We verified that the effect of SF

on DOX accumulation was concentration- and time-dependent.

The effect on P-gp expression was in accordance with obtained

increases in DOX accumulation. We showed that the decrease in

P-gp expression correlates with the strongest inhibition of P-gp

activity after 72 h of SF application. Therefore, we confirmed the

P-gp modifying effect of SF in two different MDR cancer cell lines.

SF Chemo-sensitizes MDR Cancer CellsSince SF significantly inhibits P-gp activity and expression, we

decided to combine SF and DOX with the expectation that SF

would improve DOX anti-cancer action. The effects of 5 mM SF

on the chemo-sensitivity of MDR cancer cell lines were assessed by

the MTT assay. We chose 5 mM SF as relevant concentration,

which considerably decreased the percentage of P-gp positive cells

in both MDR cancer cell lines. In order to distinguish the possible

synergistic interaction of DOX and SF from chemo-sensitizing

effect of SF, we compared the effects of simultaneous and

subsequent treatment (Table 1). The IC50 value for DOX

decreased in NCI-H460/R cells from 3.250 mM to only

3.036 mM in simultaneous treatment with SF. However, SF

promoted significant reversal of resistance to DOX (7.26-fold) in

subsequent treatment by decreasing the IC50 value for DOX from

7.083 mM to 0.976 mM (Table 1). SF also achieved significant

chemo-sensitizing effect in U87-TxR cells in subsequent treatment

promoting 17.01-fold reversal of DOX resistance (Table 1). The

decrease in IC50 value for DOX in simultaneous treatment with

SF (from 0.224 mM to 0.098 mM) was not significant in U87-TxR

cells. We confirmed the chemo-sensitizing effect of SF in

subsequent treatment with DOX. However, the improvement of

DOX effect in simultaneous treatment with SF was negligible. In

previous study, we showed that SF decreased the expression of

topo IIa, which is the main target of DOX [5]. It implies that the

effect of these two drugs in combination should be antagonistic.

Therefore, the synergy between SF and DOX observed in

subsequent treatment is obviously the result of SF facilitated

DOX accumulation and the chemo-sensitization achieved by SF

could not be merely the consequence of SF and DOX intracellular

interaction.

In this manner, we showed that SF significantly enhanced DOX

cytotoxicity in subsequent treatment. Our results suggest that the

improvement of DOX cytotoxicity was caused by SF induced

inhibition of P-gp expression and activity. In favor to this are also

the evidences that purine analogs could alter membrane glyco-

protein synthesis [34].

SF Modulates the Intracellular Pool of VEGF165 in MDRCells

Vascular endothelial growth factor (VEGF) mediates pro-

angiogenic effects. VEGF expression has been found to signifi-

cantly correlate with new vessel formation and tumor progression

in patients with NSCLC [35], while gliomas are reputed for their

high micro-vascular proliferation [36]. New targeting strategies are

aimed to block neo-angiogenesis in these two malignancies [37],

[38].

Since it was shown that 6-thioguanine has anti-angiogenic

potential [4], we decided to examine the effect of SF on gene

expression and protein secretion of VEGF165 in resistant NCI-

H460/R and U87-TxR cells. Additionally, VEGF and P-gp

expression are regulated by the same transcriptional factor –

hypoxia inducible factor-1a (HIF-1a) [39], and high concentra-

tions of VEGF could decrease the expression of P-gp [40], [41].

We examined the changes in vegf165 mRNA expression levels in

NCI-H460/R (Figure 7A) and U87-TxR cells (Figure 7B) treated

with 5 and 10 mM. The reduction of vegf165 mRNA expression in

NCI-H460/R cells achieved with 5 and 10 mM SF was 22% and

41%, respectively (Figure 7A). SF also significantly decreased

vegf165 expression in U87-TxR cells treated with 5 and 10 mM by

48% and 45%, respectively (Figure 7B).

We also assessed the VEGF expression by flow-cytometry

(Figure 7C, D, E, F). We used direct PE-labeled antibody for

VEGF to study the changes in protein expression level. Significant

decreases in mean VEGF fluorescence of NCI-H460/R and U87-

TxR cells treated with 10 mM SF were observed after 24 h and

48 h, respectively (Figure 7C, D). The decrease of VEGF

expression was transient. The best effect was illustrated by flow-

cytometric profiled histogram (Figure 7E, F). The fluorescence in

SF treated samples declined in comparison to untreated controls of

NCI-H460/R and U87-TxR by 22.5% and 51.6%, respectively.

The secretion of VEGF165 protein was examined in cell culture

medium (Figure 7G, H). The culture medium was collected from

untreated and treated samples with the same amount of cells as

well as when the amount of cells in treated samples decreased due

to SF action.

The secretion of VEGF165 after 24 h, 48 h and 72 h by

untreated U87-TxR cells (4620, 6070 and 8020 pg/ml, respec-

tively) exceeded the secretion obtained by untreated NCI-H460/R

cells (359, 845 and 1459 pg/ml, respectively). Secretion of

VEGF165 was highly stimulated (1220, 2323 and 2629 pg/ml,

respectively) when NCI-H460/R cells were treated with 10 mM SF

(Figure 7G, grey bars). However, the level of secreted VEGF165

decreased significantly after 72 h from 1459 pg/ml in control

samples to 973 pg/ml in SF treated samples if the results were not

normalized (Figure 7G, black bars). U87-TxR cells also signifi-

cantly elevated the level of secreted VEGF165 after 10 mM SF

treatment, but only at 24 h (Figure 7H, grey bars). Contrary, when

the levels of secretion were not normalized, significant decreases in

detected VEGF165 were observed after 48 h and 72 h of SF

treatment (Figure 7H, black bars). This could be the consequence

of SF anti-proliferative and apoptotic effect as well as of significant

decrease of VEGF expression observed in U87-TxR cells after

48 h of SF treatment.

According to obtained results, SF modulates the secretion of

VEGF and reduces its expression and the synthesis of correspond-

ing mRNA. Therefore, we assume that SF exerts its anti-

angiogenic potential by empting the intracellular pool of VEGF

and preventing the renewal of its synthesis. However, the detected

elevation of secreted VEGF in medium could be the consequence

of VEGF leaking from dying cells. This is supported by the fact

that the level of VEGF in medium was actually decreased along

with the decrease in number of viable cells after SF treatment.

Further experiments, particularly in co-culture with endothelial

cells, are required to clarify this point.

ConclusionsAchieving an effective treatment of lung cancer is difficult, in

particular in advanced stage diagnosed disease [42]. Physiological

CNS barriers that prevent penetration of systemically delivered

molecules limit treatment of brain malignancies [43]. Further-

more, resistance to standard chemotherapeutic agents presents

major challenge in cancer treatment. MDR phenotype allows a



cancer cell exposed to a single agent to become simultaneously

resistant to both that drug and to other drugs of unrelated

structure or function. P-gp, which is often involved in development

of MDR, is responsible for the unidirectional efflux of drugs from

cancer cells [44].

We showed that SF anti-cancer effects in resistant NSCLC and

glioblastoma cell lines include depletion of GSH, reversion of

MDR through inhibition of P-gp expression and activity, and

modulation of VEGF intracellular pool. This mechanistic study

explains SF action through modification of redox status and HIF-

1a regulation and rationalize the use of SF alone or in

combination with conventional anti-cancer agents. By this means,

SF should be considered as drug that disturbs diverse mechanisms

involved in cancer progression, beneficial for combination with

other chemotherapeutics, particularly substrates for P-gp. Since SF

inhibits HIF-1a-induced transcription of target genes important

for chemoresistance and metastasis such as mdr1 and vegf, our

results may be useful for translation in clinic, especially for

targeting solid tumors with hypoxic regions or highly-angiogenic

tumors.

Author Contributions

Revised critically the article for important intellectual content: LR SR.

Conceived and designed the experiments: MD AI MP. Performed the

experiments: MD AI AMI APR TS ZM MP. Analyzed the data: MP.

Wrote the paper: MP.

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